Naohiko Koshikawa

Expression of Trypsin by Epithelial Cells of Various Tissues, Leukocytes, and Neurons in Human and Mouse

It has long been believed that trypsin is normally synthesized only in the pancreas. In the present study, expression of trypsin in human and mouse nonpancreatic tissues was examined. Northern blot analysis of normal human tissues indicated that the trypsin gene is expressed at high levels in the pancreas and spleen and considerably in the small intestine. However, in situ hybridization and immunohistochemistry demonstrated that trypsin is widely expressed in epithelial cells of the skin, esophagus, stomach, small intestine, lung, kidney, liver, and extrahepatic bile duct, as well as splenic and neuronal cells. In the spleen, trypsin message was detected in macrophages, monocytes, and lymphocytes in the white pulp. In the brain, it was detected in the nerve cells of the hippocampus and cerebral cortex. Analysis by gelatin zymography confirmed the presence of a latent or an active form of trypsin in various normal mouse tissues. Reverse transcription-polymerase chain reaction analysis also confirmed the expression of trypsin genes in the spleen, liver, kidney, and brain of normal mice. Such a broad distribution of trypsin suggests its general roles in the maintenance of normal epithelial cell functions, the immune defense system, and the central nervous system.

Expression of gelatinase A, tissue inhibitor of metalloproteinases-2, matrilysin, and trypsin(ogen) in lung neoplasms: An immunohistochemical study

Lung cancer is a heterogeneous tumor in terms of clinical and biological behavior, and its aggressiveness depends on its invasive and metastatic properties. Matrix metalloproteinases and serine proteinases are believed to play a crucial role in invasion and metastasis of malignant tumor cells. In the present study, the authors evaluated immunohistochemically the expression of gelatinase A; tissue inhibitor of metalloproteinases-2 (TIMP-2), an inhibitor of gelatinase A; matrilysin; and trypsin(ogen) in 67 lung tumors from a variety of histological types including 17 squamous cell carcinomas, 16 adenocarcinomas, 15 small cell carcinomas, and 12 carcinoids. Interestingly, normal bronchial, bronchiolar, and alveolar epithelial cells expressed gelatinase A, TIMP-2, matrilysin, and trypsin(ogen) at varying frequencies and intensities. Bronchial smooth muscle cells and cartilage cells expressed gelatinase A alone, whereas endothelial cells, fibroblasts, and macrophages expressed gelatinase A and TIMP-2. Gelatinase A was expressed at high levels in most lung tumors examined (47% to 80%). TIMP-2 was also expressed at high levels except in the small cell carcinomas, which showed TIMP-2 expression at a lower frequency (60%) compared with other types of lung tumors (80% to 100%). Although matrilysin was expressed by tumor cells of all the histological types at various frequencies (13% to 63%), its expression was most common in adenocarcinomas. Expression of trypsin(ogen) was observed almost exclusively in adenocarcinomas (56%); other types of lung tumors expressed trypsin(ogen) far less frequently (0% to 12%). The present results, taken together with those of previous studies, suggest that gelatinase A is associated with malignant behavior of all the types of lung tumors, whereas its activity may be controlled by the endogenous inhibitor TIMP-2. The aggressive clinical behavior of small cell carcinoma may be attributable, at least in part, to a loss of the inhibitory effect of TIMP-2, as a significant proportion of these tumors showed negative or low levels of TIMP-2 expression. Matrilysin and trypsin(ogen) expressions are unlikely to be correlated with the aggressiveness of lung tumors. The expression of trypsin (ogen) may rather reflect the differentiation of adenocarcinoma cells toward normal airway epithelial cells.

Wide Distribution of Laminin-5 γ2 Chain in Basement Membranes of Various Human Tissues

Laminin 5 (LN5), a heterotrimer of laminin α3, β3, and γ2 chains, is a laminin isoform which strongly promotes adhesion, migration, and scattering of cells through binding to integrins α3β1, α6β1 and α6β4. To get an insight into the physiological functions of LN5, we prepared a mouse monoclonal antibody to human laminin γ2 chain and used it for immunohistochemical analysis of laminin γ2 chain in normal human tissues. The basement membranes of various epithelial tissues, such as the skin, lung, small intestine, stomach, kidney and prostate, were immunostained with the anti-laminin γ2 chain monoclonal antibody. In addition, the basement membrane of the surface germinal epithelium in the ovary was also positive for laminin γ2 chain. These results suggest general roles of LN5 in the anchorage of various types of epithelial cells to the underlying basement membrane and in the expression of their cellular functions. Moreover, deposition of laminin γ2 chain around small arteries and veins was observed in the thymus and spleen. This lymphatic organ-specific expression of vascular LN5 might provide a novel function of LN5 in immune responses.

Expression of trypsin in vascular endothelial cells.

Proteinases produced by vascular endothelial cells are expected to play important roles in many biological processes. Here we report that human vascular endothelial cells express trypsinogen‐2 mRNA and its protein product in culture. The trypsinogen production was stimulated by a tumor promoter and associated with cell growth. In situ hybridization analysis showed that the trypsinogen gene was significantly expressed in vascular endothelial cells around gastric tumors and in patients with disseminated intravascular coagulation (DIC). These results suggest the possible roles of endothelial cell‐derived trypsin in tumor angiogenesis and abnormal blood coagulation.

Matrilysin-specific antisense oligonucleotide inhibits liver metastasis of human colon cancer cells in a nude mouse model

Human colon cancer frequently develops liver metastasis. Matrilysin (MMP‐7), the smallest member of the matrix metalloproteinase (MMP) family, is commonly produced by human colon carcinoma cells and has been suggested to be involved in the progression and metastasis of this type of cancer. In the present study, we tested the effect of a matrilysin‐specific antisense phosphorothioate oligonucleotide on liver metastasis of the human colon carcinoma cell line WiDr in nude mice. In culture, the antisense oligonucleotide moderately inhibited the secretion of matrilysin by WiDr cells. Injection of WiDr cells into the spleen of nude mice produced many metastatic tumor nodules in the liver. When the antisense oligonucleotide was injected daily into the mice for 11 days, the formation of the metastatic tumor nodules was strongly inhibited in a dose‐dependent manner. An inhibition of liver metastasis of over 70% was obtained at a dose of 120 μg of the oligonucleotide per mouse. The antisense oligonucleotide did not inhibit tumor growth in spleen and in liver. A scrambled control oligonucleotide had no effect on liver metastasis of WiDr cells. Our results demonstrate an important role of matrilysin in liver metastasis of human colon cancer and the therapeutic potential of matrilysin antisense oligonucleotides for the prevention of metastasis. Int. J. Cancer 76:812–816, 1998.© 1998 Wiley‐Liss, Inc.

Expression of matrilysin in vascular endothelial cells adjacent to matrilysin-producing tumors

Matrilysin is believed to play important roles in tumor progression and metastasis. In the present study, we analyzed matrilysin‐producing cells in various human cancer tissues by immunohistochemistry and in situ hybridization. Tumor cells in colorectal carcinomas, pancreatic carcinomas, transitional‐cell carcinomas of the kidney and small‐cell lung carcinomas were frequently positive for matrilysin. In addition, we found that endothelial cells of arterioles and venules adjacent to matrilysin‐positive tumors expressed matrilysin mRNA and protein. The endothelial cells adjacent to matrilysin‐negative tumors and those in normal tissues were negative for matrilysin. Furthermore, analyses by casein zymography, Western blotting and RT‐PCR showed that matrilysin was weakly expressed by cultured human umbilical vein endothelial cells. Our results suggest that the expression of matrilysin in vascular endothelial cells and in tumor cells may be regulated by common soluble factors, and that endothelial cell‐derived matrilysin may contribute to tumor angiogenesis and tumor metastasis. Int. J. Cancer 72:441–445, 1997.

Production of trypsins by human gastric cancer cells correlates with their malignant phenotype

Proteolytic degradation of extracellular matrix is a critical step in tumour invasion and metastasis. To examine the role of trypsin in tumour dissemination, we cloned two variants (S4 and R3 cells) from STKM-1, a trypsinogen 1-producing diffuse gastric cancer cell line. Western blot analysis with antitrypsin antibody showed that 26 and 24 kDa proteins were highly detected in S4 conditioned medium (CM) in comparison to R3 CM. In addition to the 26 and 24 kDa proteins, 25 and 23 kDa bands, which correspond to enterokinase-activated trypsin, were found only in S4 CM. When the CMs of the two clones were treated with enterokinase, the 25 and 23 kDa trypsin activities in S4 CM were effectively increased as compared with R3 CM. When the two clones were inoculated intraperitoneally (i.p.) into nude mice, S4 cells strongly invaded the liver, pancreas and peritoneum and killed the hosts more rapidly than R3 cells: the 50% survival time was 50 days for S4 and 82 days for R3 cells. These results suggest that trypsin production is associated with the invasive growth of STKM-1 gastric cancer cells.

Stimulation of cellular growth and adhesion to fibronectin and vitronectin in culture and tumorigenicity in nude mice by overexpression of trypsinogen in human gastric cancer cells

It has previously been reported that the trypsinogen gene is expressed in various human cancers. To inves-tigate the possible role of trypsin in tumor malignancy, trypsinogen-1 cDNA was introduced into the human gastric carcinoma cell line MKN-1. The overexpression of trypsinogen-1 in MKN-1 cells stimulated cellular growth and adhesion to fibronectin and vitronectin when the trypsinogen activator enterokinase was added into the culture. Enterokinase treatment of the conditioned medium of the MKN-1 transfectants partially converted the proforms of gelatinases B and A to their apparent active forms. When the MKN-1 transfec-tants expressing trypsinogen-1 were intraperitoneally transplanted into nude mice, the mice frequently produced tumors in the colon, spleen and liver. However, the mice implanted with control MKN-1 cells produced no tumors. These results strongly suggest that tumor-derived trypsin contributes to the dissemi-nated growth of some types of cancer cells including gastric cancer. ©Lippincott Williams & Wilkins

Matrilysin gene expression in sporadic and familial colorectal adenomas

We examined the expression of matrilysin mRNA in sporadic and hereditary colorectal adenomas to clarify the role of matrilysin in tumorigenesis. Matrilysin mRNA was not detected in normal colorectal mucosa from patients with either sporadic or familial adenomas. Matrilysin mRNA expression in sporadic adenomas correlated with the degree of dysplasia and the size of the mass, whereas most of the adenomas in patients with familial adenomatous polyposis coli expressed matrilysin mRNA irrespective of adenoma size or degree of dysplasia. Because matrilysin is more likely to be expressed in adenomas with a potential for malignancy, this enzyme may play a role in the malignant conversion of colorectal adenomas. Mol. Carcinog. 19:225–229, 1997.

Sole expression of laminin γ2 chain in invading tumor cells and its association with stromal fibrosis in lung adenocarcinomas

Laminin‐5 (LN‐5), an important basement membrane (BM) protein consisting of laminin α3, β3 and γ2 chains, has been suggested to be involved in tumor cell invasion and tissue repair. In this study, the distribution of the LN‐5 subunits in atypical adenomatous hyperplasia (AAH)and different types of adenocarcinomas of the lung was examined by immunohistochemical analysis. In AAH and non‐sclerosing, well‐differentiated adenocarcinomas, the LN γ2 chain was frequently detected along with the continuous BMs. These BMs were also positive for both LNα3 and β3 chains, suggesting that LN‐5 had been deposited. In contrast, the cytoplasmic staining for the LNγ2 chain was frequently observed in tumor cells of sclerosing, well‐differentiated adenocarcinomas, as well as of moderately and poorly differentiated adenocarcinomas, without any evidence of co‐expression of the LNα3 and β3 chains. This staining pattern of the LNγ2 chain was prominent in carcinoma cells invading into interstitial stroma and was associated with the formation of a central scar in the tumor tissues. These results suggest that the LNγ2 chain monomer could be an important indicator of progression of lung adenocarcinoma.